By the end of 2007, the U.S. produced 6.5 billion gallons of ethanol per year from 139 dry grind corn plants, a 32% increase in production from the previous year (RFA (2008) Ethanol industry outlook. Renewable Fuels Association: Washington, D.C.). In the dry grind process, corn is ground and slurried to approximately 32% solids (wb). The slurry is incubated at 105° C. for several minutes to gelatinize starch and at 90° C. with amylases to break down hydrated starch to dextrins. Yeast, urea and glucoamylases are added to this mash which undergoes simultaneous saccharification and fermentation (SSF) whereby yeast anaerobically convert sugars to ethanol. Ethanol is removed during distillation and remaining solids are dewatered and dried to produce distillers dried grains and solubles (DDGS).
Though yeast productivity is dependent on a number of environmental and process factors, dry grind operations are designed with substrate and end-product inhibition of yeast in mind. The inherent release of glucose during cooking requires initial slurry solids ≦32% (w/w). This limit results from two factors: controlling mash viscosity after cooking and keeping glucose concentrations below inhibitory levels (typically 15 to 16% (w/v) (Thatipamala, et al. (1992) Biotechnol. Bioeng. 40:289-297). A granular starch hydrolyzing enzyme (GSHE) digests raw starch to glucose at ≦48° C. By gradually liquefying starch during fermentation, GSHE renders starch cooking unnecessary and avoids viscous- and inhibitory-based solids content limits imposed by glucose. The consolidated process is called simultaneous liquefaction saccharification and fermentation (SLSF) (FIG. 1A). It has been reported that using GSHE resulted in comparable yields to conventional enzymes and methods at 25% solids (Wang et al (2007) Cereal Chem. 84:10-14). GSHE permits higher solids fermentations; however, it has not be determined whether GSHE SSF fermentations can be performed at solids >32% (w/v).
Higher solids fermentations result in higher ethanol concentrations. Early onset of ethanol inhibition caused by using higher solids reduced yield and fermentation efficiency (Wang, et al (1999) Cereal Chem. 76:82-86). Therefore, in situ removal of ethanol, using technologies such as vacuum stripping, could slow ethanol accumulation and reduce early inhibition (Ramalingham and Finn 1977, Cysewski and Wilke 1977). By applying vacuum at 6.7 kPa (28 inHg) and increasing fermentation yeast densities, 12-fold higher productivities and successful fermentations at 35% glucose (w/w) have been reported (Cysewski & Wilke (1977) Biotechnol. Bioeng. 19:1125-1143), wherein normal glucose feeds are closer to 10% (w/w). Vacuum application, however, was found to cause strict anaerobiosis (O2<8 ppm), inducing nutritional deficiencies with yeast. Sparging with oxygen was expensive while sparging with air, though cheap, required a large vacuum pump to recompress N2. Therefore, the Flashferm has been proposed (Maiorella, et al (1979) Rapid ethanol production via fermentation. Rep. No. 10219. Univ. Calif. Lawrence Berkeley Lab: Berkeley, Calif.), wherein beer is pumped continuously between the fermenter at atmospheric pressure and a flash vessel. Vacuum (6.7 kPa) is applied to the flash vessel instead of the main fermenter vessel. With this strategy the fermenter can be sparged with air without requiring a large vacuum compressor.
Processes developed by Ramalingham & Finn ((1977) Biotechnol. Bioeng. 19:583-589), Cysewski & Wilke ((1977) supra), and Maiorella, et al. ((1979) supra) use a glucose feedstock and continuous fermentation whereas most modern dry grind plants use ground corn and batch fermentations. If higher solids are anticipated, circulating beer to a flash vessel may pose difficulties with pumping the beer. Furthermore, modern technologies have overcome sparging requirements. While older yeast strains require air sparging for lipid production (Andreasen & Stier (1953) J. Cell. Physiol. 41:23-36), newer strains contain sufficient lipids for batch fermentation (D'amore & Stewart (1987) Enzyme Microb. Technol. 9:322-330; You, et al. (2003) Appl. Environ. Microbiol. 69:1499-1503).